Project Summary Eukaryotic chromosome function and segregation depend on the centromere, which is the critical noncoding locus upon which the kinetochore is assembled. Human centromeres are composed of tandem arrays of ~171 bp ?-satellite DNAs, which span up to several megabases. It has been previously shown that homologous recombination at the centromere is suppressed, but the mechanism and functional significance of this suppression remain unclear. In cancer cells, chromosome translocations often occur at centromeres and adjacent pericentromeric heterochromatin, generating fusion chromosomes and frequently genetic abnormalities. It is widely accepted?though mechanistically not established?that heterochromatin, whose signatures are histone H3 tri-methylation at Lys9 (H3K9me3) and DNA methylation, suppresses homologous recombination. However, active centromeric chromatin contains the H3 variant CENP-A and does not form heterochromatin. Therefore, a still-unknown, heterochromatin-independent mechanism must exist to suppress recombination at the centromere. Interestingly, emerging evidence indicates that DNA methylation may contribute to this phenomenon; in particular, centromere instability is a cytogenetic hallmark of Immunodeficiency-centromeric instability-facial anomalies syndrome (ICF), which is caused by mutations in regulators of de novo DNA methylation, such as DNMT3b and HELLS. So far this ?centromere instability? phenotype has only been observed at the juxtacentromeric heterochromatin of chromosomes 1, 9, 16, which do not contain ?-satellites. It is not known if these ICF genes also contribute to the stability of ?-satellite DNA arrays at the active centromere. The long-term goal of this project is to reveal the overarching mechanism that supports integrity of all centromeres and suppresses rearrangements of centromere-associated repetitive sequences. For this purpose, Chromosome Orientation Fluorescence In Situ Hybridization (CO-FISH) will be employed to detect sister chromatid exchange at the centromere, and quantitative FISH (qFISH), detection of extrachromosomal circular DNA and karyotype analysis will be used to assess rearrangement at the centromere. Our recent published data indicate that CENP-A and CCAN (constitutive centromere-associated network) proteins prevent centromere rearrangements, and this functionality is compromised in cancer cell lines and in primary cells undergoing senescence. Combining these methods, we will examine the mechanism by which CENP-A, CCAN proteins and proteins related to ICF syndrome prevent centromeric rearrangements. Altogether, the outcomes of this proposal will form a novel conceptual framework of the mechanisms that maintain integrity of human centromere-associated repetitive DNAs.